The goal of this project is to investigate the molecular basis of variation in the pattern and level of alcohol dehydrogenase (Adh) expression among the eight melanogaster subgroup species of Drosophila. The proposed work is both descriptive and experimental. In the descriptive phase, variation in the developmental profiles and tissue-distribution of ADH level and in the alcohol tolerance/utilization of larvae and adults will be analyzed for all eight species. A detailed analysis of the melanogaster/simulans species pair at the ADH-protein and ADH-RNA levels will be made in order to further investigate previous reports of a marked difference in the pattern of Adh expression. The experimental phase will utilize P element mediated transformation in order to determine the molecular basis of the melanogaster/simulans difference. First, reciprocal transfer of an Adh-containing DNA fragment between ADH-negative strains of each species will establish the extent to which the interspecific difference in Adh expression is due to nucleotide substitutions in the Adh region itself as opposed to background genotype differences. The precise molecular basis of any effect that maps to the Adh fragment will be determined by in vitro recombination and site-directed mutagenesis. Transformant stocks will also be analyzed to determine the contribution of variation in the Adh region to interspecific variation in alcohol tolerance/utilization. This strategy provides a direct approach to investigating the relative importance of structural versus regulatory variants in adaptive evolutionary change.